In histology many different stains and variations thereof are utilized. For example, in hematology, several different variations of a histologic Romanowsky staining are used for differentiation of blood cell types in a blood film smeared on a glass slide. In particular, two distinct types of stains known as Wright Giemsa (WG) and May Grunwald (MG) are used. Also, May Grunwald mixture is often used in combination with Giemsa stain producing May Grunwald Giemsa (MGG) formulation. All types of Romanowsky stain formulations are prepared from two main dyes, Eosin Y and Methylene Blue using a variety of processes, each one resulting in a distinct stain formulation
Wright stain is a mixture of eosinates (Eosin Y dyes) of polychromed Methylene Blue. May Grunwald stain is a German equivalent of Jenner stain, which is a Methylene Blue eosinate similar to Wright stain but differing in not using polychromed Methylene Blue. Polychromed Methylene Blue is an alkaline solution of Methylene Blue that undergoes progressive oxidative demethylation with aging (ripening) to produce forms of all the tri, di, mono and non-methyl intermediates, resulting in a mixture of methylene blue, azures, thionine and methylene violet.
When applied to stain peripheral blood films, any type of Romanowsky stain will, in general, produce a similar type of cell coloration. However, small but important differences in stain formulation will allow for differentiating of specific conditions in the blood cells such as, for example, toxic granulation in neutrophils. If the stain formulation is not optimal (e.g., lacks sufficient fraction of pure Methylene Blue in proportion to its derivatives), the normal neutrophilic granules tend to overstain and look like toxic granules. The optimal stain composition also enhances the staining of nucleoli and polychromatophilic RBCs (reticulocytes). Therefore, it is important to maintain and tightly control the specific composition of any given stain formulation as achieved in optimization processes.
Because compositions of Romanowsky type stains are very similar, it is difficult, if not impossible, to discriminate between any (WG, MG or MGG) closely related stains and/or variations of the same stain by visual inspection or through the use of common laboratory instrumentation, such as measurement of stain pH.
In automated histology e.g., as performed by automated histological or hematological slide preparation devices, various stains may be used depending on the particular assay to be performed or user preferences. Automated slide preparation devices, regardless of whether a particular device uses a single stain or multiple stains, requires a human user to load the stain(s). As such, the stains may be inadvertently misidentified and the wrong stain may be loaded into the device. In addition, stains may arrive misidentified, unbeknownst to the device user, due, e.g., to accidental mislabeling or improper preparation. Shelf storage under inadequate conditions may affect stain composition, not noticeable by visual inspection.
Stain protocols are highly dependent on use of the proper stain. Thus, when a misidentified or incorrectly prepared stain is used, the procedure will likely produce an undesired result, e.g., sub-optimal coloration. While stain identity may be conclusively determined by complex analytical methods used to determine stain composition, e.g., high performance liquid chromatography (HPLC), these methods are arduous, consuming significant time and resources. HPLC also requires a set of reference analytes to identify peaks (bands) on a chromatogram which might be difficult to obtain for such a complex mixture as a polychomed Methylene Blue.
Methylene blue is a cationic dye (i.e., it produces positively charged ions or cations) which, in its pure form, exhibits two major absorption bands at 293 nm (π-π* transition) and 664 nm (n-π* transition) in aqueous solutions, with a 664 nm (producing blue color) band having a shoulder at 610 nm corresponding to the 0-1 vibronic transition. The specifics of absorption depend on a number of factors, including protonation, adsorption to other materials, and metachromasy—the formation of dimers and higher-order aggregates depending on concentration and other interactions. In turn, Eosin Y is an acidic dye (ionizes in solution to produce negatively charged ions or anions) which exhibits a major absorption band at 524 nm (producing red color).
Therefore, optical absorbance of a dye molecule is sensitive to modifications which the dye molecule may undergo in the process of stain preparation.